Interpretive Summary: Plants are subject to a large number of diseases, and many of these are caused by viruses. Identifying new viruses that cause diseases can be very difficult. Most plant virus tests require significant amounts of research before the proper assay can be developed. Very few assays are capable of detecting novel or emerging plant viruses, and the few assays that are useful for discovery provide very little information about these novel viruses. This paper describes a new approach for virus discovery using high throughput technology and the genetics of viruses. Plants often respond to viruses by chewing up the genetic material of the invader. These chewed up nucleic acids end up in a pool of degraded products called “small RNAs”. The novel approach determines the sequence of these “small RNAs” and uses computer technology to reassemble the small bits into the near complete viral genome. This technique was used to identify a novel virus of citrus called Citrus leprosis virus cytoplasmic type 2, a significant problem for citrus production in Brazil and a potential threat to US citrus production. The major advantage to the novel approach requires no previous research to discover and identify new viruses.

Technical Abstract:
The identification of novel plant viruses is a tricky matter. Most plant virus diagnostics are based on immunological or nucleic acid based assays, where prior characterization of the virus (either antibodies or genetic sequence) is required for reagent production. There are no universal nucleic acid signatures that can be used to amplify products from every plant virus family. General plant virus family primers are available for some of the more well studied families. However, there are multiple plant virus families for which general primers cannot be defined and even in cases where such primers exist, only a small portion of the genome can be recovered. This paper describes the adaptation and utilization of a novel next generation sequencing technique for the discovery of a new plant virus, Citrus leprosis virus cytoplasmic type 2 (CiLV-C2). The technique takes advantage of a natural plant defense response, gene silencing. When a plant host encounters a RNA virus the host will frequently attempt to disrupt infection by degrading viral RNAs using a response known as RNAi. The end product of this response is a pool of small RNAs (15 to 35 nucleotides in length) made up of degraded viral RNAs plus degraded host RNAs. To determine the sequence of CiLV-C2 small RNAs were extracted from infected plants along with healthy controls. The small RNAs were sequenced using a specific next generation sequencing technique, and bioinformatic tools were used to assemble a near complete genome sequence. The potential utility for novel virus discovery and identification of causal agents is also discussed.